Virus infections of the liver continue to cause substantial human morbidity and mortality worldwide despite advances in vaccines and antiviral therapy. Hepatitis A virus (HAV), a positive-strand RNA virus classified within the genus Hepatovirus of the family Picornaviridae, is a common cause of acute hepatitis and a Category B NIAID Priority Pathogen. Our previously published research reveals that HAV infects the liver in an extraordinarily stealthy fashion, evoking little type l/lll interferon (IFN)-stimulated gene expression despite robust replication within hepatocytes over a period of several weeks. The onset of acute hepatic injury 3-4 weeks after infection is marked by impressive increases in pro-inflammatory cytokines simultaneous with the initial appearance of virus-specific antibodies and a predominantly CD4+ T cell response that correlates with resolution ofthe infection. Our goal in this project is to better understand how HAV is recognized by the innate immune system and how this may contribute to ultimate control ofthe infection. The proposed studies build on our recent discovery that HAV, classically considered a non-enveloped virus, is released from infected hepatocytes and circulates in the blood of infected humans completely enveloped in host membranes. These novel 'enveloped HAV virions (eHAV) are fully infectious but completely resistant to neutralizing antibodies. Our preliminary data show that gradient fractions containing purified eHAV uniquely induce human pDCs to secrete IFN-a and stimulate pro-inflammatory cytokine and NLRPS inflammasomedependent IL-1B expression by THP-1 cells, while highly concentrated, standard, non-enveloped virions do not. In Aim 1, we will determine host factor requirements for activation of pDCs when co-cultured with HAV infected cells, and the RNA and protein composition of pDC-activating microvesicles in collaboration with Core B. We will also quantify pDC recruitment to the liver in hepatitis A. Aim 2 will study how eHAV is sensed by THP-1 cells and primary human monocyte/macrophages, and in collaboration with Projects 2 and 3 identify the origin of signals leading to inflammasome activation by HAV. These studies will exploit unique capabilities of Core C to provide purified recombinant NLRs for quantitative in vitro studies of their potential as direct receptors of pathogen-associated molecular patterns. Aim 3 will define innate immune responses and the molecular basis of inflammasome activation by HAV in hepatocytes. These studies support the goals of this multi-project U19 application by leveraging unique biochemical capabilities to investigate the role of NLRs as novel pathogen recognition receptors (PRRs), applying cutting-edge quantitative proteomic approaches to identify paradigm-shifting signaling pathways involved in sensing and control of human viruses, examining cross-talk between PRR pathways in the human response to a NIAID Category B Priority Pathogen, investigating intracellular trafficking of a viral ligand to the site of engagment by a PRR, and taking maximal advantage of opportunities to validate experimental findings with primary human materials.